Green Synthesis of Oxoquinoline-1(2H)-Carboxamide as Antiproliferative and Antioxidant Agents: An Experimental and In-Silico Approach to High Altitude Related Disorders

At high altitudes, drops in oxygen concentration result in the creation of reactive oxygen and nitrogen species (RONS), which cause a variety of health concerns. We addressed these health concerns and reported the synthesis, characterization, and biological activities of a series of 10 oxoquinolines. N-Aryl-7-hydroxy-4-methyl-2-oxoquinoline-1(2H)carboxamides (5a–j) were accessed in two steps under ultrasonicated irradiation, as per the reported method. The anticancer activity was tested at 10 µM against a total of 5 dozen cancer cell lines obtained from nine distinct panels, as per the National Cancer Institute (NCI US) protocol. The compounds 5a (TK-10 (renal cancer); %GI = 82.90) and 5j (CCRF-CEM (Leukemia); %GI = 58.61) showed the most promising anticancer activity. Compound 5a also demonstrated promising DPPH free radical scavenging activity with an IC50 value of 14.16 ± 0.42 µM. The epidermal growth factor receptor (EGFR) and carbonic anhydrase (CA), two prospective cancer inhibitor targets, were used in the molecular docking studies. Molecular docking studies of ligand 5a (docking score = −8.839) against the active site of EGFR revealed two H-bond interactions with the residues Asp855 and Thr854, whereas ligand 5a (docking = −5.337) interacted with three H-bond with the residues Gln92, Gln67, and Thr200 against the active site CA. The reported compounds exhibited significant anticancer and antioxidant activities, as well as displayed significant inhibition against cancer targets, EGFR and CA, in the molecular docking studies. The current discovery may aid in the development of novel compounds for the treatment of cancer and oxidative stress, and other high altitude-related disorders.


Introduction
At high altitude, the concentration of oxygen decreases, resulting in the formation of reactive oxygen and nitrogen species (RONS) [1]. High altitude RONS production causes lipid, protein, and DNA damage, as well as weakening of the enzymatic and non-enzymatic antioxidant systems [2]. Excessive ROS production in particular has been linked to the development and augmentation of cardiovascular, pulmonary, neurodegenerative, and metabolic disorders, as well as cancer [3][4][5][6][7]. Furthermore, several ailments, including cancer, Alzheimer's, and diabetes, as well as motion sickness, may occur as a result of high-altitude oxidative stress [8,9]. Aerobic respiration, apoptosis, cell growth and survival, nucleic acid synthesis, and oxidative stress are all controlled by mitochondrial signaling. Mitochondrial malfunction and the resulting mishandling of the reactive oxygen species (ROS) cascade have been linked to cancer progression [10,11]. Cancer, on the other hand, is one of the terrifying diseases that frighten scientists and researchers all over the world. In 2018, 18.1 million individuals were diagnosed with cancer, and 9.6 million died as a result of it; these numbers are expected to nearly double by 2040 [12]. Oxidative stress can potentially cause malignant cell growth [13].
The term "Green Chemistry" was coined in 1991 to describe the removal or reduction of hazardous compounds with the purpose of limiting chemical exposure to individuals and the environment [14]. As our globe faces the environmental concerns of the twenty-first century, the demand for green chemistry for the synthesis of compounds with therapeutic relevance is increasing. Green chemistry has been used for advanced technologies to minimize hazardous, unwanted waste, and environmental effects. Because of the advantages of green chemistry, such as reduced waste and cost, not only pharmaceutical companies but also other chemical industries, have begun to take steps toward it [15]. In today's world, synthetic chemists in academia and industry are continually challenged to think of more environmentally friendly ways to generate the desired target molecules [16]. Ultrasoundmediated synthesis is a solution-based green synthesis, which offers a high conversion rate in a short amount of time. The ultrasound technique increased the reaction rate even under milder conditions as compared to traditional heating methods; it is also an effective energy-saving strategy [14,[17][18][19]. Ultrasound causes acoustic cavitation during chemical reactions. High pressure (18,000 atomic pressures) and temperature (2000-5000 K) are generated by acoustic cavitation, which influence chemical transformations [20,21]. The use of ultrasonication in synthesis has grown in popularity in recent years [22][23][24][25][26].
Solvents have attracted a lot of attention in the context of green chemistry since they are a major source of contamination in organic chemical processes. As a result of the increasing environmental contamination caused by the exponential usage of volatile and poisonous organic solvents in chemical industries, chemists have been pushed to focus on alternative green solvents [27]. Solvent-free synthesis can help to reduce the dangers of volatile and poisonous organic solvents, although this is not a solution. Running a reaction in a solvent, on the other hand, is often desirable to enable mass and heat transfer. Furthermore, the correct solvent selection affects reaction rates, selectivity, and the location of chemical equilibria [28]. After all, water has numerous advantages because it is a lowcost, readily available, non-toxic, and non-flammable solvent that is appealing from both an economic and an environmental aspect [28]. Using water as a solvent, several research groups successfully synthesized quinoline, a bicyclic heterocycle [23,[29][30][31]. We discussed the ultrasonic synthesis of quinolines in green solvent water, as well as their biological activity, in the current study.
Quinoline is an important scaffold found in many anticancer drugs, including bosutinib, topiranib, pelitinib, neratinib, and others [32][33][34]. Some of the quinolines based anticancer agents and EGFR inhibitors and the target ligands (5a-j) are shown in Figure 1. The molecular docking scores of the title compounds (5a-j) were found to be greater than −7.944 Kcal/mol, indicating their efficient binding against the active site of epidermal growth factor receptor (EGFR), one of the most appealing targets for anticancer agents. Three types of interactions (H-bond, π-π-Stacking and Halogen bond) were observed in molecular docking simulation. Some of the ligands showed H-bond interaction of phenolic function with the residue Met793. Many biomarkers are triggered as a result of oxidative stress, which induces inflammation and the transformation of normal cells into malignant cells. Some of the target compounds with promising anticancer activity were also evaluated for antioxidant activity. Similarly, the carbonic anhydrase (CA) isozyme IX in some of the cancer cases promotes the proliferation and metastasis of tumors; it has also been observed to be overexpressed in many malignancies due to hypoxia [35,36]. As a result, CA could also be a promising anticancer treatment strategy. Some of the oxoquinolines bind to the CA very efficiently and display three sorts of interactions (H-bond, π-π-stacking and halogen bond).

Synthesis
7-Hydroxy-4-methyl-2H-chromen-2-one (3) was initially prepared by ultrasonication from an equimolar mixture of resorcinol (0.05 mol; 5.505 g) and ethyl acetoacetate (0.05 mol; 6.505 g~6.5 mL) in 10 mL tube adding a catalytic amount of anhydrous FeCl 3 (0.0025 mol; 41 mg) for 20 min while immersed in cooling bath to obtain a precipitate as per the reported method [37,38]. The synthetic protocol is summarized in Scheme 1. In the subsequent step, an equimolar mixture of 7-hydroxy-4-methyl-2H-chromen-2-one (3) (1 mmol; 176 mg) and substituted phenyl urea (4a-j) (1 mmol) in 10 mL water was ultrasonicated for 20-25 min (20 KHz; 130 W) and the final precipitate was separated by vacuum filtration followed by washing with water and re-crystallized from absolute ethanol to obtain the target title compounds (5a-j). The substituted phenyl urea (4a-j) was prepared as per the reported synthetic protocol [39]. The synthetic protocol is summarized in Scheme 2. The synthetic strategy investigated here has the advantages of being easy to implement and having a high conversion rate with swift reaction times in green solvent under ultrasound irradiation, as seen in our previous work [40]. Infrared (IR), nuclear magnetic resonance ( 1 H and 13 C-NMR), and mass spectral data were used to confirm the structure of the final compounds (5a-j).

Optimization of Reaction Conditions
The reaction conditions were optimized in various solvents, prior to synthesis of compound 5a, in order to choose a suitable solvent. An equimolar amount of compound 3 (1 mmol; 176 mg) and phenyl urea 4a (1 mmol; 136 mg) was subjected to ultrasound irradiation in different solvents and the yields were analyzed. The results of optimization of reaction condition are given in Table 1. The yields of the compound were found to be less in ethylacetate (34%; entry 1), acetonitrile (34%; entry 6), dioxane (36%; entry 5), toluene (44%; entry 4), and dimethyl sulfoxide (49%; entry 8). The yields were increased in the solvents like dichloromethane (59%; entry 1), ethanol (64%; entry 2), glacial acetic acid (65%; entry 9) and methanol (69%; entry 3). Except for yield variations, ultrasound-mediated synthesis was found to be uniform in all solvents. The solvent-free synthesis was found to be interesting (71%; entry 10) but it required an elevated temperature (200 • C) and 60 min to complete the reaction. The reaction time was increased to 4 h when the reaction mixture was refluxed in water, but the yield was found to be advantageous (82%; entry 11). Furthermore, when the same reaction was carried out in water under ultrasonication, the yield (91%; entry12) was increased, while the reaction time was lowered to 20 min. Table 1. Optimization of reaction conditions for the synthesis of N-phenyl-7-hydroxy-4-methyl-2oxoquinoline-1(2H)carboxamide (5a).

Exploration of Methodology
As a result, we investigated and compared the scope of this synthetic method under optimize conditions in both at the elevated temperature (200 • C) in solvent free conditions, as well as under ultrasonicated modes in water as a green solvent. The physical constants and comparative yields are given in Table 2. The ultrasound-mediated reaction was found to be more beneficial than conventional heating. Table 2. Physical constants and yields of the prepared oxoquinolines (5a-j).

Antioxidant Activity
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals (FR) scavenging activity was performed for some of the target compounds to evaluated antioxidant activity, as per reported protocol [46]. The compound 5a showed the most promising antioxidant activity with an IC 50 value of 14.16 ± 0.42 µM comparable to that of the standard ascorbic acid followed by the compound 5d with an IC 50 value of 22.90 ± 0.89 µM. The rest of the three compounds (5b, 5f, 5g, 5h and 5j) showed moderate antioxidant activity with IC 50 values ranging between 24.18 ± 0.41 and 54.45 ± 0.95 µM. The antioxidant activity of five compounds is shown in Table 4.

Molecular Docking
The molecular docking studies of ligands 5a-j were carried out against EGFR (PDB ID: 3W2R) to identify the putative mechanism of action of these ligands against cancer [47,48]. The ligands showed different types of interaction the amino acid residues within the active site of EGFR include H-bond, π-π-stacking, π-cationic and halogen bond interaction, as shown in Figure 3. The various types of interaction with the amino acid residues are shown in Table 5. The ligands 5a and 5j were found to have significant antiproliferative activity. The ligand 5a showed a H-bond with the residue Met793 with the phenolic function of coumarin ring, while π-π-stacking was observed between the N-phenyl ring and the residues Leu788 and Ala743 (Figures 4 and 5). The ligand 5j showed two H-bonds, one between the phenolic function of coumarin and the residue Asp855 and another between the carbonyl function (of amide) and the residue Thr854. The ligand 5j also displayed a π-π-stacking (of phenyl ring) with the residue Leu788 and a halogen bond interaction of 3-chloro function with the residue Asp855 (Figures 4 and 5). The docking images of the rest of the compounds are given in Figures S1 and S2 (Supplementary Materials).     Similarly, CA was found to be linked with the proliferation of some of the cancer cells due to hypoxia [35,36]. Since the compounds showed anticancer as well as antioxidant activities, docking against CA isozyme IX (PDB ID: 3DC3) was also performed and the docking scores are given in Table S1 (Supplementary Materials) [49]. The ligands showed different types of interaction with the amino acid residues within the active site of CA include H-bond, π-π-stacking, π-cationic and halogen bond interaction. The ligand 5a showed efficient binding (docking score = −5.337) and displayed three H-bond interations with residues Gln67 (with phenolic function of coumarin), Gln92 (with carbonyl function) and Thr200 (with NH function, through the water molecule). Similarly, the ligand 5h (docking score = −5.643) displayed two H-bond interations with residues Gln67 (with phenolic function of coumarin), and Gln92 (with carbonyl function). The 2D binding interactions of ligands 5a and 5h are shown in Figure 6, while the 2D binding interaction of ligand 5g is shown in Figure S3

ADME Studies
ADME studies are necessary for a drug's successful absorption from the gastrointestinal system, as well as its oral bioavailability [50][51][52][53][54]. The physicochemical and pharmacokinetic parameters were studied by freely available ADME software [50]. All the compound 5a-j displayed a permissible number of H-bond acceptors (<10), H-bond donors (<5), rotatable bonds (<10) and log P (<5), as shown in Table 6. None of the compounds violated the Lipinski rule of 5, making them promising agents [55]. The most promising compounds (5a and 5j) displayed promising physicochemical and pharmacokinetic parameters in ADME prediction studies. These compounds (5a and 5j) displayed high gastrointestinal (GI) absorption (as seen in the radar plots) as well as their ability to cross the blood-brain barrier (BBB) (as seen in boiled egg diagram) (as shown in Figure 7) together with zero pan assay interference compounds (PAINS) prediction [56]. Table 6. The ADME studies of compounds 5a-j.

Discussion
Quinolines are a class of bicyclic hetero-aromatic compound with a wide range of biological activities. Many of the anticancer quinolines are in clinical trials or are in the early stages of development [32]. In the current study, a green approach was used for the synthesis of oxoquinolines (5a-j). All of the quinoline analogues followed the Lipinski rule of five and ADME predictions, indicating that they could be used as oral drugs. There were three to four numbers of rotatable bonds, making the compound flexible enough to bind with molecular target. Compounds 5a and 5j were discovered to be capable of crossing the blood-brain barrier, as shown in the boiled egg representations and bioavailability radar plots (Figure 7). However, none of the compounds were recognized as PAINS materials in SWISS ADME prediction studies. All these compounds were synthesized using a green approach. We attempted the synthesis of oxoquinolines in several solvents, with the best results obtained in water. Water has various advantages because it is a lowcost, readily available, non-toxic, and non-flammable solvent that is both economically and environmentally acceptable [28]. Using water as a solvent, several research groups successfully synthesized quinoline, a bicyclic heterocycle [23,[29][30][31]. Similarly, when compared to conventional synthesis, ultrasound-mediated synthesis is consistently found to be superior in terms of yields and reaction times [23,37].
The antiproliferative activity of the compounds (5a-j) was carried out at 10 µM on nearly five dozen cancer cell lines and the results were represented as GP as well as %GIs.
Compound 5a also demonstrated promising DPPH free radical scavenging activity with an IC 50 value of 14.16 ± 0.42 µM, while compound 5d showed antioxidant activity with and IC 50 value of 22.90 ± 0.89 µM and rest of the compounds showed moderate antioxidant activity. Antioxidant supplementation appears to be an essential step in preventing or reducing oxidative damage associated with high altitude exposure [57,58]. The literature survey revealed that acetazolamide (a specific inhibitor of carbonic anhydrase), is used to treat high altitude pulmonary oedema (HAPE) [59,60]. The ligands 5a, 5g, and 5h efficiently bind within the active site of CA.

Antiproliferative Activity
The target compounds (5a-m) were tested at 10 µM for their antiproliferative activity against nine different panels of 60 cancer cell lines. It was measured at the National Cancer Institute using the Sulforhodamine B (SRB) assay (NCI US) [42][43][44][45].

Antioxidant Activity
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals (FR) scavenging activity was carried out according to the standard protocol reported to evaluate the antioxidant activity of the some of the target compounds [46].

Molecular Docking Studies
The molecular docking against EGFR was performed for the ligands, 5a-j. The EGFR (PDB: 3W2R) X-ray crystal structure with a resolution of 2.05 Å; R-value 0.220 (observed) was obtained from the protein data bank [47]. The ligands 5a-j were saved as a mol file and the docking was done as per the reported protocol [48].
Similarly, the molecular docking against CA was performed for the ligands, 5a-j. The CA (PDB: 3DC3) X-ray crystal structure with a resolution of 1.70 Å; R-value 0.141 (work) was obtained from the protein data bank [49]. The ligands 5a-j were saved as a mol file and the docking was done as per the reported protocol [35].

ADME Studies
The pharmacokinetic and physicochemical parameters were studied by freely available ADME prediction software [50]. The various parameters include number of H-bond donors, number of H-bond acceptors, number of rotatable bonds, lipophilicity, bioavailability, GI absorption, BBB permeation, PAINS alert and P-pg substrate.

Conclusions
Following ultrasound irradiation, we successfully prepared oxoquiolines (5a-j) in high yield in green solvent water. All of these compounds adhered to the Lipinski rule of five, as well as physicochemical and pharmacokinetic parameters as measured by ADME software. The antiproliferative activity was carried out against 60 NCI cancer cell lines at 10 µM. Compounds 5a and 5j demonstrated the most promising activity against TK-10 (82.90% growth inhibition) and CCRF-CEM (58.61% growth inhibition) cell lines respectively. Compound 5d also demonstrated promising DPPH free radical scavenging activity with an IC 50 value of 14.16 ± 0.42 µM. Three types of interactions were observed in molecular docking studies: H-bond, π-π-stacking, and halogen bond interactions. The reported work could be expanded through molecular modification, which would be extremely beneficial to researchers working on anticancer research programs. Since the compounds showed significant anticancer and antioxidant activities, the title compounds were further studied for their CA inhibitory activity. Some of the ligands (5a, 5g, and 5h) displayed efficient binding with CA in the in-silico studies. The antioxidant activity of compounds 5a and 5h were found to be promising.
Some of the compounds displayed promising anticancer and antioxidant activities. Furthermore, all the ligands showed efficient binding within the active sites of EGFR, while some of the ligands efficiently bind within the active site of the CA in-silico studies. Antioxidant supplementation appears to be an essential step in preventing or reducing oxidative damage associated with high altitude exposure. As a result, the current discovery could aid in the development of novel therapeutic compounds for the treatment of cancer, oxidative stress, and other high-altitude disorders.
Supplementary Materials: Supplementary materials containing characterization of compounds (5a-j) are available online; Figure S1: The 2D interactions of ligands within the active site of EGFR; Figure S2: The 3D interactions of ligands within the active site of EGFR; Figure S3: The 3D interactions of ligand 5g within the active site of CA IX; Table S1: The molecular docking results of compounds 5a-j against CA IX (PDB ID: 3DC3).